Method of cell culture

ABSTRACT

A method for the culture of cells of the type which grow in films and substantially in monolayers, the method being intended to be employed for virus production, the cells to be cultivated being fixed on porous silica spherules which are maintained in suspension in the culture medium.

United States Patent [1 1 Bizzini et al.

[ 1 Feb. 20, 1973 METHOD OF CELL CULTURE Inventors: Bernard Bizzini, 18,rue Ernest Delosion, Neuilly; Jean-Claude Chermann, 14 rue du PrieureSaint- Nicolas, Villepreux; Claude Jasmin, 10, rue Mesnil, Paris; MarcelRaynaud, 115, boulevard de la Reine, Versailles, all of France Filed:July 2, 1970 Appl. No.: 52,062

Foreign Application Priority Data July 9, 1969 France ..6923294 US. Cl..195/1.7, 195/1.1 Int. Cl. ..C12k 9/00 Field of Search ..424/89;l95/1.l, 1.7, 1.8;

[56] References Cited UNITED STATES PATENTS 3,493,651 2/1970 Sloane..424/89 OTHER PUBLICATIONS Willmer, Cells and Tissues in Culture;Methods, Biology and Phsiology, Vol. I, published by Academic Press, NewYork, 1965, page 40.

Primary Examiner-Richard L. Huff Attorney-Craig, Antonelli & Hill [57]ABSTRACT 2 Claims, No Drawings METHOD or CELL CULTURE This inventionrelates to a method for the culture of cells of the type which grow infilms and substantially in monolayers, this method being intended to beemployed for virus production.

The culture of cells in monolayer films is well known and has permittedvery rapid development of virological techniques and the manufacture ofanti-viral vaccines from viruses produced in vitro under reproducibleand readily controllable conditions.

However, culture in stationary bottles or flasks permits the use of onlya small surface area relative to the volume of the flasks and to thequantity of culture medium which is employed. In a non-agitated flask,the cells develop in a monolayer film on the bottom of the flask.

It has been possible to achieve an improvement by carrying out theculture of cells in cylindrical tubes carried by a wheel which is drivenin slow motion. By means of this method, the culture medium is agitatedcontinuously and the cells develop over the entire surface of the tube,thereby permitting maximum utilization of the culture medium. In orderto increase the surface area afforded for the multiplication of cells toan even greater extent, it has been proposed more recently to make useof particles of certain organic polymers which are maintained insuspension in a culture medium by moderate agitation. Thus, Van Wezelhas employed spherical particles of DEAE Sephadex A 50 which aremaintained in suspension in a culture medium.

However, the last-mentioned technique carries disadvantages by reason ofthe toxicity of some of the particles employed (Sephadex is toxic abovea limiting concentration of 1 to 2 g/l), thereby entailing the need tocover the particles with collodion prior to use. Moreover, the cellsadhere very strongly to the surface of the particles and it proves verydifficult to detach them. Treatment with trypsin permits the recovery ofscarcely more than percent of the cells, the majority of which remainsattached to the particles of Sephadex.

Sephadex, which is made up of polyglucoses (dextran gel), cannot readilywithstand temperatures which are higher than 110 C without incurring thedanger of decomposition and this impedes sterilization.

The method of cell culture as contemplated by the invention overcomesthe different disadvantages referred-to in the foregoing.

In accordance with this method, the cells to be cultivated are fixed onporous mineral particles which are maintained in suspension in a culturemedium.

In general, the most suitable porous mineral particles are sphericalparticles having shapes which facilitate recovery of cells and whichhave the effect as far as possible of preventing losses of materialarising from even moderate agitation.

It is readily apparent that the nutrient media which are employed inbiological cultures are aqueous solutions and that the porous mineralparticles employed must consequently be insoluble therein and must inaddition be inactive with respect to the useful products which aredissolved in said nutrient media.

In accordance with the invention, use is advantageously made of poroussilica spherules which are available industrially and can have widelydifferent textural and structural properties. The spherules are known bythe trade name Spherosil and exist in a series of forms which differfrom each other in their porosity. The size range of these spherules isbetween 125 and 150 microns. The different types which are suitable foruse are characterized by the mean pore diameter.

Type Mean pore diameter XOAAOO A XOA.20O A XOB.075 320 A X08030 760 AXOB.0l5 1,350 A The quantity of silica spherules employed is usuallywithin the range of l to 10 g per liter of culture medi- Surprisingly,it has been observed that, after inoculation of the nutrient medium withsuitable strains, the cell cultures develop while progressively coveringthe surfaces of the silica spherules and while even forming bridgesbetween some of these latter. The cells can readily be recovered aftersettling by stopping the agitation and by rapid trypsinization.

The underlying reason for the advantages arising from the use of poroussilica spherules in cell cultures is incompletely understood. However,it is considered that the porosity of the spherules acts favorably notonly in order to reduce the density of said spherules but also byfacilitating the exchanges of solutions since it has been found thatglass spherules are not capable of producing equally satisfactoryresults.

One example of cell culture on porous silica spherules will now bedescribed without any limitation being implied and solely in order toprovide an explanatory illustration of the invention.

EXAMPLE The strict minimum of EAGLE is employed as culture medium, towhich a proportion of 10 percent of decomplementized calf serum is addedfor a period of 30 minutes at 56 C, and which contains per milliliter 50units of penicillin and 50 units of kanamycin. The pH of this medium isadjusted to 7 by means of 55 g/l sodium bicarbonate solution. Thismedium is placed in a proportion of 500 ml per flask in a number offlasks each having a capacity of 100 ml and each provided with twolateral openings for withdrawing or adding liquid and a central plug inwhich is held a wire for gripping a magnetic rod. Said flasks are placedin an oven with a variable-speed stirrer, the temperature of said ovenbeing maintained at 37 C. There were added in each flask 2 g of silicaspherules having a particle diameter between 125 and 150 microns and amean pore diameter of slightly less than 100 A. Said spherules werepreviously washed in the hot state with 6 N hydrochloric acid and thenwith distilled water, then in a detergent and finally rinsed withdistilled water, then double-distilled. Sterilization of said spheruleswas carried out in a dry-heat oven at a temperature of C for a period ofone hour.

Inoculation is carried out with approximately 10" cells per milliliterof nutrient medium which are taken from cultures prepared from mousethymus and infected with the Rauscher murine leukemia virus.

Growth is studied by sampling silica spherules in suspension after 24,48 and 72 hours. After 24 hours, the cells are attached to the spherulesbut do not cover the entire spherule surface; after 48 hours, then after72 hours, the entire spherule surface is covered, whereupon thespherules are grouped into masses and joined together by colonies ofcells which form bridges. A mass of spherules which floats freely in theculture medium is sometimes suspended from a cell which is attached to aspherule.

The recovery of cells is carried out after 72 hours by rapidtrypsinization after settling of the cells. Viability of the cells thusobtained as detennined by counting after staining with trypan blue isclose to 100 percent, their number being approximately 4 X permilliliter of culture medium.

It will be understood that, in industrial practice, the culture of cellson silica spherules can be carried out in accordance with a number ofdifferent processes, whether non-continuous, semi-continuous or whollycontinuous.

This method is applicable to the culture of standard cell strains ofhuman origin and more generally to the culture of all types of cellswhich grow in films. It is particularly well suited to mass cellproduction which require a substrate for multiplication and to theobtainment of large quantities of viruses in a minimum spatial volume.The method is of considerable interest in the manufacture of anti-viralvaccines from viruses produced in vitro.

What we claim is:

1. A method of culture of cells intended for virus production and of thetype which grow in a monolayer film, wherein the cells to be cultivatedare fixed on porous silica spherules, having diameters of about 125microns, which are maintained in suspension in the culture medium, themean diameter of the pores of said spherules being within the 1350A-range.

2. In a method for culture of cells intended for virus production and ofthe type which grow in a monolayer film, the improvement comprisingfixing the cells to be cultivated on porous silica spherules, which aremaintained in suspension in the culture medium, said spherules havingdiameters from about to about l50 microns and mean pore diametersbetween 100 and 1350 A.

1. A method of culture of cells intended for virus production and of thetype which grow in a monolayer film, wherein the cells to be cultivatedare fixed on porous silica spherules, having diameters of about 125microns, which are maintained in suspension in the culture medium, themean diameter of the pores of said spherules being within the 100 - 1350A-range.